Cholesterol-rich microdomains like rafts and caveolae are specialized regions of the plasma membrane and play an important role for several cellular processes e.g. signal transduction, and for the life cycle of certain viruses (e.g., the entry and exit steps). These domains are enriched in cholesterol, sphingomyelin, ganglioside GM1 and caveolin proteins. The cholesterol molecules are intercalated between the lipid acyl chains and cause a decrease of the fluidity of these membrane regions leading to their resistance against treatment with non-ionic detergents like Triton X-100. Therefore, these regions are also referred to as detergent resistant microdomains (DRMs). The specific lipid composition of DRMs leads to the selective incorporation and concentration of specific cellular proteins. Such proteins often exhibit Glycosylphosphatidylinositol (GPI) anchoring and fatty acylation.
It is known that viral proteins of certain viruses e.g. the envelope protein (Env) of the ecotropic murine leukemia virus (E-MLV) or of the human immunodeficiency virus type 1 (HIV-1) associate with DRMs after transport to the plasma membrane. Similarly, Gag proteins of HIV-1 prefer DRMs as cellular destinations after synthesis in the cytoplasm. Mutations of HIV-1 Env or E-MLV Env palmitoylation sites or the HIV-1 Gag myristoylation site impair the association of these proteins with DRMs. Knock out of Env palmitoylation sites led to a decreased viral titer due to a reduced Env incorporation into the viral particles.
Therefore, DRMs enriched in viral capsid and envelope proteins are considered to be platforms for assembly and budding of viruses from infected cells.
The virus envelope consists of envelope proteins (Env) embedded in a membrane consisting of a lipid double layer, whereas the viral membrane composition resembles the lipid composition of DRMs and differs from the average distribution of lipids in the plasma membrane of the host cell.
It is also known that the viral envelope may also contain cellular proteins picked up from the virus producing cell during the budding process. This has been used to incorporate specific membrane proteins e.g. complement inhibitors like CD59 (WO 00/17374) into envelops of viral particles making use of the natural transport pathway of the host cell.
Recently such approaches have also been used to increase the immunogenicity of simian immunodeficiency virus (SIV) based virus-like particles (VLPs) by addition of CD40 ligand and granulocyte-macrophage colony-stimulating factor (GM-CSF) or to modify murine leukaemia virus (MLV) based VLPs with cytokines such as IL-4 to induce differentiation of monocytes, or IL-2 to induce proliferation of peripheral blood mononuclear cells (PBMCs).
Modification of retroviral vectors so far depends on the genetic engineering of virus producer cell lines by transfection. Such an approach is very inconvenient and time consuming, because for each component to be inserted into viral envelops the respective host cell has to be genetically modified. Such an approach is also limited to proteins and polypeptides which have to be synthesized by the host cell and transported to the plasma membrane.
A typical cellular transport pathway makes use of a Glycosylphosphatidylinositol (GPI) anchor to direct proteins and polypeptides to the outer leaflet of cell membranes. Glycosylphosphatidylinositol (GPI) anchors are attached to protein precursors at the endoplasmatic reticulum (ER) membrane by a transamidase enzyme complex and delivered to the outer leaflet of the plasma membrane. GPI-linked proteins serve different functions i.e. in the regulation of complement activity (CD59; CD55) or as hydrolytic enzymes (e.g. alkaline phosphatase and renal dipeptidase). GPI anchors consist of a hydrophilic oligosaccharide and a lipophilic fatty acid part.
It was already demonstrated that the lipophilic part of said GPI anchors may mediate the exogenous insertion of proteins into lipid membranes of cells (Medof (1996); Legler (2005); McHugh (1995); Premukar (2001) and artificial lipid membranes (Ronzon (2004); Morandar (2002)) which are generally characterized by a low protein content in relation to the fatty acid content. The process of insertion of substances like proteins is termed “painting” (Medof (1996)). It has further been demonstrated that GPI anchors can be added to previously non-GPI-linked proteins (e.g. green fluorescent protein (GFP) by genetically addition of a GPI signaling sequence (GSS) to their C-terminal end and that these proteins retain their biological functions (McHugh (1995); Premukar (2001); Legler 2005).
In contrast to artificial or cellular membranes the amount of viral envelope proteins in virions is extremely high so that the lipids of the virus envelope are completely shadowed making viruses e.g. highly resistant to detergents and environmental influences. Typical lipid to protein mol fractions in cell membrane expressed as mole percentage (mol %) are 80:20 or higher whereas in typical viral envelopes the ratio is 30:70 or less (M. K. Jain, R. C. Wagner: Introduction to Biological Membranes, New York (Whiley) 1980, ISNB 0471034711). The accessibility of the virus membrane lipid layer is drastically reduced which makes it unlikely to insert voluminous proteins or other voluminous target domains into the viral envelope after release of the virus from its host cell. While trying to generate more efficient therapeutic viral vectors for gene therapy the inventors developed methods for an easier and more rapid anchoring of substances into membranes of enveloped viruses, especially in order to provide a quick and flexible alternative to engineering of genetically modified virus producing cell lines.
It was absolutely unexpected and surprising for the inventors that compounds, especially proteins linked to membrane anchor domains like Glycosylphosphatidylinositol (GPI) anchors can successfully be inserted into lipid double layers of viral envelopes when added exogenously to isolated viral particles resulting in viral particles with altered surface characteristics. The method presented herein is useful for generation of virus particles with well designed chemical and biological characteristics depending on the target domains inserted.